Biosynthetic studies using both [14C]- and [32P]-labelled substrates and a cell-free system to synthesise 1-O-alkyl moieties in glycerolipids, have shown that the three initial steps in ether-lipid biosynthesis in Leishmania mexicana promastigotes resemble those described for mammals and are associated with glycosomes. Purified glycosomes were able to sequentially synthesise the first intermediates of the ether-lipid biosynthetic pathway [acyl-dihydroxyace-tonephosphate (DHAP), alkyl-DHAP and acyl/alkyl-glycerol- 3-phosphate (G3P)] when incubated in the presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH. However, when glycosomes were incubated under the same conditions in the presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and phosphatidic acid was observed without any formation of alkyl-G3P, suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as substrate. Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP synthase activities were located inside glycosomes whereas the alkyl/acyl-DHAP oxidoreductase activity was associated with the cytoplasmic face of the glycosomal membrane. The G3P acyltransferase (G3P-AT) and lyso- phosphatidic acid acyltransferase activities were not found inside glycosomes. The results suggest that the DHAP-AT and G3P-AT activities are catalysed by two distinct enzymes associated with different sub-cellular compartments.
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